Vol. 26, No. 2

JOURNAL OF VIROLOGY, May 1978, p. 249-256

0022-538X/78/0026-0249$02.00/0 Copyright) 1978 American Society for Microbiology

Printed in U.S.A.

Purification and Characterization of Varicella-Zoster VirusInduced DNA Polymerase ENG-CHUN MAR,* YUAN-SHEN HUANG, AND ENG-SHANG HUANG Departnent ofMedicine and Cancer Research Center, School of Medicine, University of North Caroluia, Chapel Hil, North Carolna 27514 Received for publication 17 November 1977

Infection of WI-38 human fibroblasts with varicella-zoster virus led to the stimulation of host cell DNA polymerase synthesis and induction of a new virusspecific DNA polymerase. This virus-induced DNA polymerase was partially purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. This virus-induced enzyme could be distinguished from host cell enzyme by its chromatographic behavior, template specificity, and its requirement of salt for mima activity. The enzyme could efficiently use poly(dC) oligo(dG)12 18 as well as poly(dA) oligo(dT)12 18 as template-primers. It required Mg2e for m l polymerization activity and was sensitive to phosphonoacetic acid, to which host a- and ,B-DNA polymerases were relatively resistant. In addition, this induced DNA polymerase activity was enhanced by adding 60 mM (NH4)2S04 to the reaction mixture. Varicella-zoster virus (VZV), a member of the human herpes-group viruses, is the causative agent of varicella (chickenpox) in children and of herpes zoster (shingles) in adults. Significant herpes zoster infection has been discovered in patients with malignancies such as lymphoma and Hodgkin's disease (20). As a matter of fact, the occurrence of herpes zoster infection generally results from the activation of endogenous VZV already present in the individual in a latent state (7). viviruses, several Vlherpes-group vlruses, Among the herpes-group rus-induced DNA polymerases have been found in herpes simplex virus (HSV)-infected baby hamster kidney cells and WI-38 cells (14, 26), human cytomegalovirus (HCMV)-infected human fibroblasts (8), Marek's disease virus-infected duck embryo fibroblasts (2), and EpsteinBarr virus-transformed lymphoblastoid cells (17). 19) demonstrated (5, 19) two reports reports (5, Recently, two demonstrated a virus-specific of inducing is capable that VZV deoxypyrimidine kinase in VZV-infected human lung cells. In view of the finding that the replication of VZV is sensitive to phosphonoacetic acid (PAA) (16), and the fact that virus-induced DNA polymerases universally exist in various herpesvirus-infected cells, we made an attempt to determine if a virus-induced DNA polymerase also exists in iV VZV-infected cells. also that a virus-induced DNA We report here nected polymerase also exists in the VZV system. The purification and characterization of the VZVinduced DNA polymerase, as well as of host cell DNA polymerases, are also described.

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MATERIALS AND METHODS

Cels and viru8. The WI-38 strain of human fibroblasts (Hayflick), obtained from the American Type Culture Collection (ATCC; CCL-75), was used for the

entire study. The cells (passages 20 to 28) were subcultured in minimal essential medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 10% fetal calf serum, 100 U of penicillin, and 100 pg of streptomycin per ml. For maintenance, 4% fetal serum was used. VZV used in this study was isolated from a theinurban who wasatinfected during 3-year-old child of chickenpox prevalence N.C., ChapelinHill, and WI-38 was propagated 1976. The virus cellsApril identified by neutralization and complement fixation tests. Virus of passages 8 to 11 was used for this study. Preparation of enzyme extracts. Huang's procedure (8) for obtaining the enzyme extracts from infected cells was modified as follows. Eight roller bottles of confluent WI-38 cells were trypsinized and infected with VZV by co-cultivation with VZV-infected cells at a 4:1 ratio. Seventy-two hours after when more than 90% of cells showed cytoinfection, the cels were washed twice with icepathic effect, chilled TBS (0.05 M Tris-hydrochloride [pH 7.4]-0.15 M NaCl) and once with hypotonic buffer A (0.05 M Tris-hydrochloride [pH 7.81-0.001 M MgCl20.001 M dithiothreitol [D¶TT). The infected cells were scraped off with the aid of a rubber policeman and were suspended in hypotonic buffer A with 0.25% Nonidet P.40. After hypotonic treatment for 20 min, the suscentrifuged in a Sorvall HB4 rotor at pension was 2,000 rpm for 10 min. The supernatant fluid was pooled and saved. The pellet was resuspended in 4 ml of buffer B (0.02 M Tris-hydrochloride [pH 7.8]-0.1 M NaCl-0.015 M MgCl2r0.008 M DTT) and sonically treated in an ice bath for 3 min with a Branson Sonifier cell disruptor. This sonically treated suspension and the supemnatant fluid were combined and centrifuged

249

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J. VIROL.

MAR, HUANG, AND HUANG

in a Spinco SW27 rotor at 25,000 rpm for 2 h at 20C to remove cellular organelles and debris. The clarified supernatant cell extract was used for enzyme purification. Mock-infected extract was also prepared simultaneously from the same amount of mock-infected stationary cultures (eight roller bottles). Noninfected logphase cell extracts were obtained from 70% confluent log-phase cultures in an identical manner. DEAE-celiulose column chromatography. The cell extracts were loaded onto a DEAE-cellulose column (1.6 by 15 cm, DE-52; Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) pre-equilibrated with buffer D (0.05 M Tris-hydrochloride [pH 7.8]-0.001 M EDTA-0.001 M DTT-5% glycerol). The column was washed with 20 ml of buffer D and then 40 ml of buffer D containing 0.05 M NaCl. A linear gradient of 0.1 to 0.7 M NaCl in buffer D was then applied to the column. Fractions of 1.5 ml were collected, and a 50Ad volume from each fraction was assayed for DNA polymerase activity with activated calf thymus DNA as the template. The fractions of the DNA polymerase activity peak eluted at a low salt concentration (0.05 M NaCl), and containing,B-DNA polymerase, were pooled and frozen in 50% glycerol. A second DNA polymerase activity peak, eluted at a concentration of 0.15 to 0.25 M NaCl, was collected and subjected to further purification.

Phosphocellulose chromatography. The proce-

dure previously described in the HCMV-induced DNA polymerase system was used with slight modification, as follows. A phosphocellulose column (1.6 by 8 cm, Whatman P-1l) was pre-equilibrated with buffer D

containing 0.1 M NaCl. Bovine serum albumin (fraction V, 5 mg in 5 ml of buffer D) was first applied to the column to enhance the recovery of enzyme and eliminate nonspecific binding. After the column was washed with 30 ml of 0.1 M NaCl in buffer D, the pool of the second DNA polymerase peak derived from the

DEAE-cellulose was diluted to a concentration of less than 0.1 M NaCl and applied to the phosphocellulose column. Buffer D (10 ml) with 0.1 M NaCl was used for washing after the sample was applied. The elution was carried out with a gradient of 0.1 to 0.5 M NaCl in buffer D. Fractions of 1.5 ml were collected, and samples (50 id) from each fraction were assayed for DNA polymerase activity with activated calf thymus DNA, as well as poly(dA) - oligo(dT)12-18, as template-primers in the presence or absence of 0.06 M (NH4)2SO4. DNA polymerase assay. The DNA polymerase assays, as described previously (8), are based on measurement of the incorporation of tritium-labeled nucleoside triphosphate into an acid-precipitable pro uct. In system A, with activated calf-thymus DNA as the template, the stock reaction mixture (2.5x concentration) contained: Tris-hydrochloride (pH 7.8), 0.1 M; MgCI2, 0.025 M; DTT, 0.001 M; bovine serum albumin, 1.25 mg/ml; dATP, dCTP, dGTP, 0.25 mM each; and TTP, 0.025 mM. For the assay, 0.1 ml of the stock reaction mixture, 0.1 ml of activated calf thymus DNA (200 pg/ml), 0.05 ml (or as stated) of enzyme, and 1 !ACi of [3H]TTP (48 Ci/mmol) were incubated at 370C for 1 h. The reaction was terminated by adding 2 ml of chilled 20% trichloroacetic acid. The precipitated material was collected on glass fiber filter paper (GF/C

Whatman) or a nitrocellulose membrane filter (B-6; Schleicher & Schuell Co., Keene, N.H.) and washed with 5% trichloroacetic acid by filtration. After drying, the filters were counted in liquid scintillation fluid. In system B, with synthetic poly(dA) oligo(dT)1218, poly(rA) -oligo(dT) 2 l8, or oligo(dT)12.18 as the template-primer, the reaction mixture contained the same ingredients as in system A, except that dATP, dCTP, and dGTP were omitted. The synthetic templateprimers in the amount of 2 ,ug/0.1-ml reaction were used instead of activated calf thymus DNA. In system C, with synthetic poly(dC) oligo(dG)121s or poly(rC) * oligo(dG)121s as the template-primer, only one substrate, dGTP (0.025 mM for 2.5x concentration), together with 1 ,uCi of [3H]dGTP (specific activity, 42 Ci/mmol), was used; the template-primer concentration was 2 ug/reaction. Reagents and chemicals.PAA was obtained from Bodmen Chemical, Richmond, Va.; p-hydroxymercuribenzoate, 1,10-phenanthroline, DTT, calf thymus DNA, and bovine serum albumin (fraction V) were purchased from Sigma Chemical Co., St. Louis, Mo. DEAE-cellulose (DE52) and phosphocellulose (p-il) were obtained from Reeve-Angel (Whatman Biochemicals Ltd.). Synthetic polynucleotide-oligodeoxynucleotides were purchased from P-L Biochemicals, and [3H]TTP and [3H]dGTP were obtained from New England Nuclear Corp., Boston, Mass. 1,7-Phenan-

throline, a gift of J. Harrison; was from Alfrea, Bader Chemicals.

RESULTS

DNA-dependent DNA polymerase activity in VZV-induced and mock-infected human fibroblasts. By using DEAE-cellulose column chromatography, two main peaks with DNA polymerizing activity were detected in VZV-infected cells (Fig. 1). From the observa-

tions of Lewi et al. (13), Srvastava (23), and Chang and Boilum (4) in human celLs and mouse L cells, the enzyme activity eluted in low salt (0.05 M NaCl) should be ,B-polymerase (25), and the second one, eluted at a concentration of 0.18 M NaCl, should have been a-polymerase plus VZV-specific DNA polymerase. The enzyme fractions from peak 2 of the DEAE-celiulose column were further chromatographed on a phosphoceulose column (Fig. 2). a virus-induced Thereduon results revealed that that the the vlrus-induced DNA polymerae could not be clearly resolved

when activated calf thymus DNA was used as the template-primer. However, if a synthetic template, e.g., poly(dA) . oligo(dT)hs18, was used,

a peak of enzyme activity at an NaCl concentration of 0.32 M was conspicuous and proved to be VZV-induced DNA polymerase, which was not found in either stationary- or log-phase, mockor hand, most ifectdm Onthe ifected systems. On the other hand, the host cell enzyme (a-polymerase) eluted at 0.24 M NaCl was easily identified simply because it could efficiently use the activated calf thymus

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Fraction Number FIG. 1. DEAE-cellulose chromatography of cell extracts from VZV- and mock-infected ir-38 cells. Combined cytoplsmic and nuclear extracts wure applied to apre-equilibrated DEAE-cellulose column. Sampel s were eluted by 0.05 M NaCL and a linear 0.1 to 0.7 M NaCl gradient in buffer D. Each frlaction (50 IL) was assayed for DNA polymerase activity with activated calf thymus DNA as the template-primer. Elution profile extracts of VZV-infected (0) and mock-infected (0) stationary-phase cells. of the enzyme activities ftom

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FIG. 2. Further chromatography of DA fr-action enzyme of infected and noninfected stationary-phase cells phosphocellulose columns. The enzyme fr-actions fr-om the second peak (D,) of DEAE-cellulose (Fig. 1, fr-actions 25 to 45) were pooled, diluted with buffer D, and applied to phosphocellulose columns. The elution was carried out with a linear gradient of 0.1 to 0.5 M NaCl in buffer D. Activated calf thymus DNA (0) and POIY(dA) -oligo(dT)12-18 (0) were used as template-primers for enzyme activity assays. Very low activity can be seen in mock-infected cells (top). In the infected-cell fr-action, peak 1 enzyme (P~) eluted at an NaCl conentatin o arun 0SP.12 M, P2 eluteat.241 M, andP was found toa^ be viu-idcenzm t,hat on

252

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MAR, HUANG, AND HUANG

DNA as a template-primer. A clear distinction between the viral DNA polymerase activity and host enzyme activity could be achieved by adding (NH4)2SO4 (60 mM) or PAA (50 ,ug/ml) to the assay system, in which viral enzyme activity was stimulated by the presence of (NH4)2SO4 but completely inhibited by PAA; host cell enzymes (a and ,B) were resistant to PAA but were suppressed almost entirely by (NH4)2SO4 (Fig.

3).

Properties of virus-induced DNA polymerase. (i) Template-primer specificities. Virus-induced enzyme obtained from phosphocellulose column chromatography, together with virus-stimulated host cell enzymes (a and f polymerases), were examined for template-primer specificities. Virus-induced DNA polymerase could efficiently use the synthetic template poly(dA) oligo(dT)12-18 as well as poly(dC) oligo(dG)12-18. Furthermore, the viral DNA polymerase was unable to use poly(rA)* oligo(dT)12 18, poly(rC) oligo(dG)12 18, or oligo(dT)12 18 as a template-primer; this implies that the virus-induced DNA polymerase is an enzyme with a character distinct from that of reverse transcriptase (1), RNA-dependent DNA polymerase from lymphoblastoid cells (13), and ter-

minal deoxynucleotidyl transferase (3, 4). The VZV-infected host enzymes (a and ,B), however, prefer activated calf thymus DNA over synthetic poly(dA) -oligo(dT)12-18 and poly(dC) -oligo(dG)12-18 (Table 1). (ii) Effect of divalent metal ions on virusinduced enzyme activity. Our results revealed that the concentration of Mg2e needed to obtain maximal polymerization was around 5 to 10 mM. Host cell a- and B-DNA polymerases are very sensitive to ammonium sulfate treatment. At a concentration >30 mM (NH4)2SO4, both a- and fl-enzymes were inhibited, whereas the virusinduced DNA polymerase activity was enhanced about twofold in the presence of 0.06 M (NH4)2SO4 (Fig. 4). This salt stimulation effect on the virus-induced enzyme was more significant when synthetic poly(dA) oligo(dT)1218 was used as the template-primer. Under such conditions, both host a- and fl-enzyme activities were totally suppressed by ammonium sulfate at a final concentration of 0.03 to 0.24 M (Fig. 4). However, a- and fl-enzymes were more resistant to (NH4)2SO4 treatment when activated calf thymus DNA was used than when poly(dA) * oligo(dT)12-18 was used as the template-primer. (IE) Effect of p-hydroxymercuriben-

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Fraction Number FIG. 3. Determination of viral DNA polymerase from host cell enzymes purified byphosphocellulose column chromatography performed after the addition of PAA (50 pg/ml) or (NH4)S04 (60mM) to the reaction mixture. Poly(dA) oligo(dT)12-18 was used as the template-primer. Viral enzyme activity was enhanced by the presence of 50 mM (NHJ2S04 but was completely suppressed by adding PAA (50 pg/ml) to the reaction mixture. Host cell enzymes were resistant to PAA but inhibited by the presence of (NH4)2SS4O

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VOL. 26, 1978

253

VZV-INDUCED DNA POLYMERASE

TABLE 1. Template-pruner specificities of VZV-induced and WI-38 host cell DNA polymerases

Assay system

A A A B B C C B

Template-primer

VZV-induced enzyme incorporated

Substrate

Activated calf thymus DNA [3H]TTP, cold dNTPa Native calf thymus DNA [3HITTP, cold dNTP Denatured calf thymus DNA [3H]TTP, cold dNTP Poly(dA) oligo(dT) s18 [3HfTTP, cold TTP [3H]TTP, cold TTP Poly(rA) oligo(dT)Mi2wb [3H]dGTP, cold dGTP Poly(dC) oligo(dG)1i1s Poly(rC) oligo(dG) sib8b [3H]dGTP, cold dGTP Oligo(dT)1i8 [3HTITP, cold TTP

(pmol) 26 3 5 62 1 106 0.7

Purification and characterization of varicella-zoster virus-induced DNA polymerase.

Vol. 26, No. 2 JOURNAL OF VIROLOGY, May 1978, p. 249-256 0022-538X/78/0026-0249$02.00/0 Copyright) 1978 American Society for Microbiology Printed i...
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